The present invention relates generally to caspases and their role in apoptosis, and in particular, to nucleic acids encoding membrane derived caspase-3, the encoded polypeptides, antibodies thereto, and methods of producing and using membrane derived caspase-3 polypeptide.
The normal physiological process of programmed cell death, also known as apoptosis, plays a critical role in the maintenance of tissue homeostasis. The apoptotic process in multicellular organisms ensures that the rate of new cell accumulation produced by cell division is offset by a commensurate rate of cell loss due to death. A typical result of apoptosis are certain morphological changes in a cell, including fragmentation of nuclear chromatin, compaction of cytoplasmic organelles, dilatation of the endoplasmic reticulum, decreased cell volume, and alterations in the plasma membrane. The end result of programmed cell death is phagocytosis of apoptotic cells and prevention of an inflammatory response. Disturbances in apoptosis that prevent or delay normal cell turnover can be just as important to the pathogenesis of diseases as are known abnormalities in the regulation of cell proliferation and the cell cycle. Similar to cell division, which is controlled by complex interactions between cell cycle regulatory proteins, apoptosis is regulated under normal circumstances by a complex network of gene products that interact to either induce or inhibit cell death.
The stimuli that regulate the function of these apoptotic gene products include both extracellular and intracellular signals. Either the presence or removal of a particular stimulus can be sufficient to evoke a positive or negative apoptotic signal. Physiological stimuli that inhibit or reduce the likelihood of apoptosis include, for example, growth factors, extracellular matrix, CD40 ligand, viral gene products, neutral amino acids, zinc, estrogen, and androgens. In contrast, stimuli that promote apoptosis include, for example, tumor necrosis factor (TNF), Fas, transforming growth factor xcex2 (TGFxcex2), neurotransmitters, growth factor withdrawal, loss of extracellular matrix attachment, intracellular calcium, and glucocorticoids. Other stimuli, including those of environmental and pathogenic origin, may also induce or inhibit apoptosis. Although diverse signals and complex interactions of cellular gene products mediate apoptosis, the results of these interactions ultimately lead into a cell death pathway that is evolutionarily conserved between humans and invertebrates.
Several gene families and products that modulate the apoptotic process have now been identified. One family is the Bcl-2 proteins, which can function to modulate apoptosis in a wide variety of cell systems (Oltvai and Korsmeyer, Cell 79:189-192, 1994; Reed, Nature 387:773-776, 1997). The over-expression of Bcl-2 has been shown to inhibit the activation of cytoplasmic caspases following apoptotic stimuli in several cell systems (Armstrong et al., J. Biol. Chem. 271:16850-16855, 1996; Chinnaiyan et al., J. Biol. Chem. 271:4573-4576, 1996; Boulakia et al., Oncogene 12:29-36, 1996; Srinivasan et al., J. Neurosci. 16:5654-60, 1996). Although Bcl-2 inhibits the onset of apoptosis, it does not impede already initiated apoptosis (McCarthy et al., J. Cell Biol. 136:215-217, 1997). Most Bcl-2 family members associate with cellular membranes, such as the mitochondrial outer membrane, the nuclear envelope, and the endoplasmic reticulum (Reed, Nature 387:773-776, 1997; Krajewski et al., Cancer Res. 53:47014714, 1993; Yang et al., J. Cell. Biol. 128:1173-1184, 1995; Lithgow et al., Cell Growth Differ 3:411-417, 1994); however, it remains unclear how the membrane bound Bcl-2 exerts control over another key set of apoptosis regulators, the soluble cytoplasmic, aspartate-specific cysteine proteases (xe2x80x9ccaspasesxe2x80x9d).
The caspase family of cysteine proteases are essential effectors of the apoptotic process (Yuan et al., Cell 75:641-652, 1993; Alnemri et al., Cell 87:171, 1996; Cohen, Biochem. 326:1-16, 1997; Miller, Semin. Immunol 9:35-49, 1997; Salvesen and Dixit, Cell 91:443-446, 1997). Caspases are synthesized as inactive zymogens, which are activated by proteolytic processing to yield large (xcx9c18 kDa) and small (xcx9c12 kDa) subunits that associate to form active enzymes (Thornberry et al., Nature 396:768-774, 1992; Nicholson et al., Nature 376:37-43, 1995; Stennicke and Salvesen, J. Biol. Chem. 272:25719-25723, 1997). Diverse apoptotic stimuli cause the activation of specific caspases which then initiate a protease cascade by proteolytically processing additional caspases (Srinivasula et al., Proc. Natl. Acad Sci USA 93:14486-14491, 1996; Yu et al., Cancer Res. 58:402-408, 1998). Once activated, these downstream (executioner) caspases kill cells by cleaving specific molecular targets that are essential for cell viability or by activating additional pro-apoptotic factors (Liu et al., Cell 89:175-184, 1997; Enari et al., Nature 391:43-50, 1998; Salvesen and Dixit, Cell 91:443446, 1997).
Caspase-3 is an example of a downstream xe2x80x9cexecutionerxe2x80x9d caspase thought to cleave a number of important cellular proteins involved in DNA replication, DNA repair, RNA splicing, protein phosphorylation, and chromosomal fragmentation during apoptosis (Cohen et al., Biochem. J. 326:1-16, 1997, Enari et al., Nature 391:43-50, 1998, Liu et al., Cell 89:175-84, 1997). This enzyme is synthesized as a 32 kDa procaspase that is processed into mature 20/17 kDa (large) and 12 kDa (small) subunits by cleavage at Asp 9, Asp 28, and Asp 175 (Fernandes-Alnemri et al., J. Biol. Chem. 269:30761-64, 1994; Tewari et al., Cell, 81:801-9, 1995; Fernandes-Alnemri et al., Proc.Natl.Acad.Sci. USA, 93:7464-69, 1996, Nicholson et al., Nature 376:37-43, 1995). Procaspase-3 can be activated by a number of proteases involved in apoptosis, including caspases-1, -8, -9, and -10, as well as the serine protease Granzyme B (Stennicke et al., J. Biol. Chem. 273:27084-90, 1998, Fernandes-Alnemri et al., Proc.Natl.Acad.Sci. USA, 93:7464-69, 1996; Quan et al., Proc Natl Acad Sci USA, 93:1972-76, 1996, Krebs et al., J. Cell Biol. 144:915-26, 1999). Immunocytochemical experiments indicate that procaspase-3 is primarily a cytoplasmic protein (Krajewski et al., Cancer Res. 57:1605-13, 1997; Posmantur et al., J. Neurochem. 68:2328-37, 1997; Chandler et al., J. Biol. Chem. 273:10815-18, 1998), while activated caspase-3-like enzyme is found in the cytoplasm and the nucleus (Martins et al., J. Biol. Chem. 272:7421-30, 1997). Additionally, it was reported that procaspase-3 may localize to the mitochondrial intermembrane space (Mancini et al., J. Cell Biol. 140:1485-95, 1998).
The dysfunction or loss of regulated apoptosis can lead to a variety of pathological disease states because apoptosis maintains tissue homeostasis in a range of physiological processes, including embryonic development, immune cell regulation and normal cellular turnover. For example, the loss of apoptosis can lead to the accumulation of self-reactive lymphocytes associated with many autoimmune diseases. Additionally, abnormal loss or inhibition of apoptosis can also lead to the accumulation of virally infected cells and hyperproliferative cells, such as neoplastic or tumor cells. Similarly, the irregular activation of apoptosis can contribute to a variety of pathological disease states, including, for example, acquired immunodeficiency syndrome (AIDS), neurodegenerative diseases, and ischemic injury. Treatments that are specifically designed to modulate the apoptotic pathways in these and other pathological conditions can change the natural progression of many of these diseases.
Thus, there exists a need to identify apoptotic genes and their gene products and methods of modulating apoptosis for the therapeutic treatment of human diseases. The present invention satisfies this need and provides related advantages as well.
The present invention provides in part the discovery of a novel membrane derived caspase-3 polypeptide or functional fragment thereof and isolated nucleic acid molecules encoding such polypeptides. The present invention also provides membrane derived caspase-3 polypeptide encoding nucleic acid molecules in vectors, host cells, gene delivery vehicles, and kits, as well as antibodies directed to naturally and recombinantly expressed membrane derived caspase-3 polypeptide. The present invention also provides methods of inducing apoptosis, of treating certain diseases, and of identifying an agent that alters the activity of membrane derived caspase-3 polypeptide.
In one aspect, the present invention provides an isolated nucleic acid molecule consisting essentially of a sequence encoding membrane derived caspase-3 polypeptide of SEQ ID NO:3. In one embodiment, the nucleic acid molecule encodes membrane derived caspase-3 polypeptide of SEQ ID NO:3 that oligomerizes with a caspase. In another embodiment, the invention provides a vector having an isolated molecule consisting essentially of a sequence encoding membrane derived caspase-3 polypeptide of SEQ ID NO:3.
In another aspect, the present invention provides an isolated nucleic acid molecule encoding a membrane derived caspase-3 consisting essentially of a single stranded or double stranded polynucleotide sequence of SEQ ID NO:2. In another embodiment, the invention provides a vector having an isolated nucleic acid molecule encoding a membrane derived caspase-3 consisting essentially of a single stranded or double stranded polynucleotide sequence of SEQ ID NO:2.
In a related embodiment, the aforementioned vectors are a viral vector. In another embodiment, a nucleic acid expression vector having any one of the aforementioned nucleic acid molecules wherein the nucleic acid molecule is operably linked to a promoter. In a further embodiment, the invention provides the nucleic acid expression vector wherein the promoter is an inducible promoter. In yet another embodiment, the aforementioned nucleic acid expression vectors are individually contained in a host cell. In a further embodiment, the invention provides a host cell containing the aforementioned nucleic acid expression vectors wherein the host cell is selected from the group consisting of a bacterium, a yeast cell, a nematode cell, an insect cell, and a mammalian cell.
In another aspect, the invention provides an isolated membrane derived caspase-3 polypeptide consisting essentially of SEQ ID NO:3. In one embodiment, the invention provides the isolated membrane derived caspase-3 polypeptide consisting essentially of SEQ ID NO:3 wherein the polypeptide oligomerizes with a caspase. In another embodiment, the aforementioned polypeptide oligomerizes with a caspase wherein the caspase is selected from the group consisting of caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, and caspase-14.
In another aspect, the invention provides an antibody specific for a membrane derived caspase-3 polypeptide, the polypeptide consisting essentially of SEQ ID NO:3. In one embodiment, the invention provides the aforementioned antibody wherein the antibody is a monoclonal antibody. In another embodiment, the invention provides a cell expressing any one of the aforementioned antibodies.
In another aspect, the invention provides an antibody that does not specifically recognize a cytoplasmic derived caspase-3 and does specifically recognize a membrane derived caspase-3 polypeptide, the membrane derived caspase-3 polypeptide consisting essentially of SEQ ID NO:3.
In another aspect, the invention provides an antibody that specifically recognizes a cytoplasmic derived caspase-3 and does not specifically recognize a membrane derived caspase-3 polypeptide, the membrane derived caspase-3 polypeptide consisting essentially of SEQ ID NO:3.
In another aspect, the invention provides a method of producing a membrane derived caspase-3 polypeptide, comprising culturing a host cell containing a nucleic acid expression vector comprising at least one promoter operaby linked to a nucleic acid molecule encoding a membrane derived caspase-3 polypeptide, the nucleic acid molecule consisting essentially of SEQ ID NO:2, under conditions and for a time sufficient for expression of the polypeptide. In one embodiment, the aforementioned method includes the nucleic acid expression vector having a promoter that is inducible. In a further embodiment, the invention provides the aforementioned methods further comprising the step of contacting the host cell with a caspase activator under conditions and for a time sufficient to activate the membrane derived caspase-3 polypeptide. In yet another embodiment, the invention provides the aforementioned methods further the host cell expressing a caspase activator under conditions and for a time sufficient to activate the membrane derived caspase-3 polypeptide. In still another embodiment, the activator of the aforementioned methods, wherein the activator is selected from the group consisting of caspase-1, caspase-8, caspase-9, caspase-10, and Granzyme B.
In another aspect, the invention provides a membrane derived caspase-3 polypeptide produced by any one of the aforementioned methods.
In another aspect, the invention provides a method of inducing apoptosis in a cell, comprising delivering to a cell an effective amount of an isolated nucleic acid molecule encoding a membrane derived caspase-3 polypeptide, the nucleic acid molecule consisting essentially of SEQ ID NO:2, under conditions and for a time sufficient for expression of the polypeptide and therefrom detecting apoptosis of the cell. In one embodiment, the aforementioned method of inducing apoptosis wherein the cell comprises a tissue culture cell. In a further embodiment, the aforementioned method of inducing apoptosis wherein the tissue culture cell is selected from the group consisting of 697 lymphoblastoid cells, E15 primary brain cortical cells, MN9D cells, Jurkat T cells, THP-1 cells, and FL5. 12 cells. In another embodiment, the aforementioned method of inducing apoptosis wherein the step of delivering to the cell is selected from the group consisting of injection, transfection, transformation, electroporation, and receptor mediated endocytosis. In yet another embodiment, the aforementioned method of inducing apoptosis wherein the step of delivering administering the nucleic acid molecule to the circulatory system of a warm-blooded mammal in which the cell is located. In still another embodiment, the aforementioned method of inducing apoptosis wherein step of apoptosis detection is selected from the group consisting of altered cellular morphology, DNA fragmentation, annexin binding, caspase activity, and mitochondrial release of cytochrome c.
In another aspect, the invention provides a method of inducing apoptosis in a cell, comprising delivering to a cell an effective amount of a membrane derived caspase-3 polypeptide, the polypeptide consisting essentially of SEQ ID NO:3, under conditions and for a time sufficient to detect therefrom the induction of apoptosis of the cell. In one embodiment, the aforementioned method of inducing apoptosis in a cell wherein the of delivering to the cell comprises injecting the polypeptide. In another embodiment, the aforementioned method of inducing apoptosis wherein step of apoptosis detection is selected from the group consisting of altered cellular morphology, DNA fragmentation, annexin binding, caspase activity, and mitochondrial release of cytochrome c.
In another aspect, the invention provides a gene delivery vehicle comprising any one of the aforementioned nucleic acid molecules wherein the nucleic acid molecule is operably linked to a promoter. In one embodiment, the aforementioned gene delivery vehicle wherein the vehicle is a retrovirus or adenovirus. In another embodiment, the aforementioned gene delivery vehicle wherein the acid molecule is associated with a polycation. In a further embodiment, the aforementioned gene delivery vehicle further comprising a ligand that binds a cell surface receptor.
In a related embodiment, the invention provides a method of treating cancer, comprising administering to a patient any one of the aforementioned gene delivery vehicles, wherein the gene delivery vehicle is internalized by tumor cells. In another embodiment, the invention provides a method of treating autoimmune disease, comprising administering to a patient any one of the aforementioned gene delivery vehicles, wherein the gene delivery vehicle is internalized by cells mediating autoimmune disease. In yet another embodiment, the invention provides a method of treating viral infections, comprising administering to a patient any one of the aforementioned gene delivery vehicles, wherein the gene delivery vehicle is internalized by virally-infected cells. In still another embodiment, the invention provides a method of treating bacterial infections, comprising administering to a patient any one of the aforementioned gene delivery vehicles, wherein the gene delivery vehicle is internalized by bacterially-infected cells.
In another aspect, the invention provides a kit for screening for agents that alter apoptosis, comprising a host cell and an isolated nucleic acid molecule consisting essentially of a sequence encoding a membrane derived caspase-3 polypeptide of SEQ ID NO:3. In one embodiment, the aforementioned kit wherein the host cell is a eukaryotic cell. In another embodiment, the aforementioned kit wherein the eukaryotic host cell is selected from the group consisting of 697 lymphoblastoid cells, E15 primary brain cortical cells, MN9D cells, Jurkat T cells, THP-1 cells, and FL5. 12 cells.
In another aspect, the invention provides a kit for screening for agents that alter apoptosis, comprising a membrane derived caspase-3 polypeptide, the polypeptide consisting essentially of SEQ ID NO:3 and a detection reagent that specifically binds to at least one of the foregoing polypeptides. In one embodiment, the aforementioned kit wherein the detection reagent is an antibody or antigen-binding fragment thereof.
In another aspect, the invention provides a composition, comprising a membrane derived caspase-3 polypeptide consisting essentially of SEQ ID NO:3, and an excipient or diluent.
In another aspect, the invention provides a method for identifying an agent that alters the activity of a membrane derived caspase-3 polypeptide consisting essentially of SEQ ID NO:3, comprising contacting the membrane derived caspase-3 polypeptide with a caspase substrate in the presence and absence of at least one candidate agent, and comparing the levels of caspase substrate turnover and therefrom identifying an agent that alters the activity of the membrane derived caspase-3 polypeptide. In one embodiment, the aforementioned method wherein the caspase substrate comprises a site cleaved by a caspase selected from the group consisting of a protein, a polypeptide, an oligopeptide, a peptide mimetic and a peptide. In another embodiment, the aforementioned methods wherein the substrate comprises the peptide DEVD. In a further embodiment, the aforementioned methods wherein the membrane derived caspase-3 polypeptide is part of a membrane fraction. In still another embodiment, the aforementioned methods wherein membrane fraction comprises membranes selected from the group consisting of heavy membranes and nuclear membranes. In yet another embodiment, the aforementioned methods wherein membrane fraction comprises a heavy membranes and nuclear membranes. In another embodiment, the aforementioned methods wherein substrate turnover is detected by time course analysis or endpoint analysis. In still another embodiment, the aforementioned methods wherein caspase substrate turnover detection is performed by a method selected from the group consisting of fluorescence spectroscopy, mass spectrometry, HPLC, colorimetry, fluorography, radiography, gel electrophoresis, chromatography and N-terminal peptide sequencing. In a further embodiment, the aforementioned methods further comprising incubating the membrane derived caspase-3 polypeptide with a caspase activator prior to or concurrent with the addition of the caspase substrate.
In another aspect, an agent identified by any one of the aforementioned methods that alters the activity of a membrane derived caspase-3 polypeptide consisting essentially of SEQ ID NO:3. In one embodiment, the aforementioned agent wherein the agent inhibits or enhances the activity of the membrane derived caspase-3 polypeptide.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.